Tissues were homogenized in cold SDS lysis buffer (50mM Tris pH7.5, 70mM urea, 250mM sucrose and 2% SDS) with protease inhibitor cocktail (Roche 04693124001) and phosphatase inhibitor cocktail II and III (Sigma P5726 and P0044). Total proteins were separated in 4–12% Invitrogen BT precast gel (NP0315) or any KD Bio-rad TGX precast gel (456–9033) and transferred to Nitrocellulose membranes. Antibodies were from Cell Signaling (α-4E-BP1(#9252), α-phospho-4EBP1 Ser 65 (#9451), α-phospho-4EBP1 Thr 37/46 (#9459), α-S6 (#2217), α-phospho-S6 Ser 235/236 (#2211), α-AKT1 (#4691), α-phospho-AKT Ser 473 (#4060), α-JNK (#9258), α-phospho-JNK Thr183/Tyr185 (#9251), α-eIF4E (#2067), α-eIF4G (#2469), α-S6K1 (#2708), α-phospho-S6K1 Thr 389 (#9234), α-PRAS40 (#2691), α-phospho-PRAS40 Ser 183 (#5936), α-IRS1 (#3015), α-phospho-IRS1 Ser 636/639 (#2388), α-HSP90 (#4877), α-αtubulin (#3873) and α-β ACTIN (#4967)), abcam (α-FGF21 (ab171941), and α-UCP1 (ab23841)), and EMD Millipore (α-DEPTOR, ABS222)
α αtubulin
Manufactured by Abcam
α-αtubulin is a protein that is a component of microtubules, which are cytoskeletal structures found in eukaryotic cells. It plays a crucial role in the formation and function of the mitotic spindle during cell division.
Automatically generated - may contain errors
Lab products found in correlation
α 4e bp1, by Cell Signaling Technology (1 mentions)
α hsp90, by Abcam (1 mentions)
α β actin, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Irdye labeled secondary antibody, by LI COR (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
α her2, by Cell Signaling Technology (1 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (1 mentions)
4 to 12 bis tris gels, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
2 protocols using α αtubulin
1
Western Blot Analysis of Cell Signaling
2
Immunoblotting for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Confluent plates of cells were harvested, washed with PBS, and lysed with cold RIPA buffer (Sigma Aldrich) with protease inhibitor and phosphatase inhibitor tablets (Sigma Aldrich). Lysates were cleared by centrifugation and protein quantified using the Thermo Fisher BCA Protein Assay protocol. Equal amounts of proteins were prepared in SDS loading buffer supplemented with β-mercaptoethanol, boiled at 95 °C to denature proteins, loaded onto precast 4 to 12% Bis–Tris gels (Life Technologies), and subjected to electrophoresis at 100 V. They were then transferred to PVDF membranes (Life Technologies) with the iBlot2 Transfer System for 7 min or 1h wet transfer at 100V. Membranes were blocked in Intercept Blocking Buffer (LICOR Biosciences) followed by incubation with indicated primary and IRDye-labeled secondary antibodies (LICOR Biosciences). Bands were visualized with the Odyssey® Imaging Systems. Primary antibodies used were as follows: α-Her-2 (Cell Signaling), α-BCAM (R&D), α-α-Tubulin (Abcam), α-Myc-tag (Upstate).
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